AMINO ACID FORMATION AND UTlLIZATION JN NEUROSPORA

Transcription

1 AMINO ACID FORMATION AND UTlLIZATION JN NEUROSPORA BY I. ANDERSSON-KOTTO, G. EHRENSVARD, G. HiiGSTRijM, I,. REIO, AND Ii:. SALUSTE (From the WenneT-Gren Institute of Experimental Biology, Departm.cnt of Physiological Chemistry, University of Stockholm, Stockholm, Sweden) (Received for publication, February 1, 1054) In order to make a detailed study of differences of amino acid formation and utilization in Neurospora crassa, wild type, compared with its lysineless mutant 4545, a series of experiments has been carried out involving the cultivation of both strains on labeled acetate, C13H3C1400Na. From the data on the utilization of the 2 carbon atoms of acetate for amino acid synthesis, the normal and the mutant strain may be compared with special regard to the over-all influence of a mutation on amino acid metabolism in general. In addition, the utilization of uniformly Ci4-labeled lysine by the two strains growing on acetate medium has been studied. The strains of Neurospora used throughout these experiments mere the wild type 1A and the lysineless mutant We are indebted to Dr. G. W. Beadle and Dr. F. J. Ryan for providing us with these strains. Experiments with Labeled Acetate The procedure used for all the experiments described here was as follows: Sterile medium in stainless steel 6 liter Kluyver flasks, containing 4 liters of culture medium, was inoculated with media from 7 day-old sucrose agar cultures. The inorganic constituents of the medium were the same as those used in previous work with Neurospora (1). The carbon sources were ammonium tartrate, 5 gm. per liter, and sodium acetate (3HeO), 5 gm. per liter. In some experiments, e.g. in the cultivation of the Zysineless mutant, 50 mg. of L-lysine monohydrochloride were added per liter of culture medium. After 18 hours of cultivation under vigorous aeration at the mycelium was removed by centrifugation and transferred to the same volume of fresh medium containing all components necessary for maximal growth. Isotope-labeled acetate, C13H&1400Na, was added as the last component, whereupon t,he air outlet of the flasks II-as immediately connected with CO, absorption units. Samples of respiratory CO*, precipitated as barium carbonate, were taken from time to time during the following 10 hour period of cultivation. After termination of the growth period, the mycelium was isolated by 455

2 456 AMINO SCIDS IN NEUROSPORA centrifugation, washed three times with distilled water, then with ethanol, and finally with ether, three times with each solvent. The dry weight of the wild type mycelium averaged 1.1 gm. per liter of culture medium. The yield of dry material from the mutant was somewhat lower, usually 0.7 to 0.8 gm. per liter. The protein content of the dry material averaged 10 per cent. The mycelial material was hydrolyzed in 6 N hydrochloric acid. The techniques of the isolation and degradation of amino acids were the same as those previously used in investigations of amino acid metabolism in other microorganisms (Z-6), with the modification that the acetaldehyde obtained from ninhydrin treatment of alanine was oxidized directly to acetic acid. This was degraded by a Schmidt reaction (7, 8) after purification on a Celite column (9). Threonine was converted by long time treatment with HI to cr-amino-n-butyric acid, which was decarboxylated by ninhydrin. The resulting propionaldehyde was oxidized with dichromate to propionic acid and the latter degraded as described by Phares (8). The amounts of alanine and threonine degraded correspond to about 0.5 mu of substance. All amino acid fragments were ultimately converted into carbon dioxide by combustion of the different degradation products, except in the cases of plain decarboxylation (ninhydrin or Schmidt reactions). The carbon dioxide obtained was trapped as barium carbonate and examined for 03 and Cl4 content. The results are presented in Table I. With regard to the Cl3 and Cl4 content in the respiratory carbon dioxide in the different experiments presented in Table I, there are two features that have their counterpart in similar experiments with Torulopsis utilis and Escherichia coli. First, the total isotope content reaches a maximum some time after the actual start of the experiment and later decreases to some extent. Secondly, the quotient CY4: CY3 during the first few minutes of the experiment is higher than 3, indicating that at this stage the liberation of Cl4 from the acetate carboxyl exceeds the corresponding output of Cl3 from the methyl moiety. Later the quotient reaches a steady value of 1.5 to 1.6. From comparison of the isotope content in the respiratory carbon dioxide from Experiment A (Table I) (acetate as the sole carbon compound in the medium) and Experiment B (acetate + tartrate) it was evident that tartrate was not utilized to any appreciable amount. The over-all distribution of Cl3 and Cl4 in different carbon atoms of amino acids isolated from the mycelial material corresponds closely to the distribution in amino acids from Torulopsis and E. coli (see Table II). The labeling in the acetate used, C13H3C1400Na, is for simplicity denoted as CZH3C*OONa, or 2-o. The labeling sequences in alanine, serine, and

3 ANDERRSOWKOTT6, EHRENSVARD, HijGSTRijM, REIO, SALUSTE 457 TABLE Comparison of Isotope Content of Respiratory Carbon Dioxide during Simultaneous Cultivation of Wild Type N. crassa (W) and Lysineless Mutant 4646 (L) Experiment A, isotope content of acetate; in COOH, 58,600 c.p.m. per mg. of C; in CH3, 11.9 atom per cent excess CP; 5.0 gm. of acetate per liter. In Experiment B, isotope content of acetate, in COOH, 47,500 c.p.m. per mg. of C; in CHZ, atom per cent excess 03; acetate and ammonium tartrate, each 5.0 gm. per liter. In both experiments the readings under Cl3 are in atom per cent excess; under C J, counts per minute per mg. of C. 03 and 04 denote isotope content in respiratory CO2 in per cent of the corresponding 03 and 04 content in acetate at the start of the experiments. The isotope content in total carbon of mycelial material after termination of the experiments was, for Experiment A, Cl3 4.20, for W; Cl3 3.66, Cl44490 for L; for Experiment B, CP3.54, Cl for W; C133.35,C for L. I Time 1 wc; L 1 w ( L 1 w y L 1 LV y L ~ T;;, min. o & o Experiment , , , ,200 18,700 18, , , ,200 20,650 23,250 20,000 19,400 19,100 Experiment I 0.26 I D.93 5,980 11,100 2,800 9, ,860 11, ,800 13, ,400 15, ,500 16, ,800 19, ,360 21) ,560 18, ,300 16, ,960 12, ,860 12, ,860 8,460 I A B

4 458 AMINO ACIDS IN NEUROSPORA aspartic acid (z-z-x: l, x-x-x l, and x O-X-X-X l ) are in accordance with what has been found for the corresponding amino acids from Torulopsis (3), E. co& (4), and regenerating rat liver (7) isolated in similar experiments. The sequences found for threonine, x o-x-x-x l, and for valine, I> TABLE Isotope Content in Different Carbon Atoms of Amino Acids Isolated from Wild Type Neurospora Neurospora cultivat,ed on C13H&Y400Na in the presence of ammonium tartrate (T) and without (A). 03 and Cl4 denote isotope content in per cent of the corresponding content of the acetate used in the experiments; see Table I, where the figures for isotope content in acetate and respiratory carbon dioxide are presented in series W. Amino acids and degradation products Glutamic acid, cr-cooh.... Aspartic 01- and &COOH.... Glycine, COOH... Alanine,... Serine, Valine, Isoleucine, COOH. Threonine, Leucine, COOH.. Lysine, Arginine, IL Histidine, Proline, Tyrosine, Phenylalanine, COOH II Isotope content of acetate C (CHa = 100) T A I- T A T 23.0 i fp COOH = 100) I A Alanine, a-carbon atom. p-carbon Serine, a-carbon p-carbon Glycine, or-carbon Threonine, a-carbon atom. o-carbon r-carbon. Valine, all carbon atoms carboxyl rsoleucine, same. Leucine, same, except

5 hnderssofkott6, EHRENSVLRD, HijGSTRijM, REIO, S.4LUSTE 459 x-x-x l, are analogous to the findings from earlier experiments with other microorganisms. A further parallel is that the leucine carboxyl is entirely C14-labeled and consequently is derived from the acetate carboxyl by a direct route. The same holds for the lysine carboxyl, a feature having its counterpart in Torulopsis, but differs from the result in E. coli. A full degradation of tyrosine from the above experiments has been reported previ- TABLE Comparison of Distribution of Isotope jrom C13HQ400Na in Amino Acids Isolated from Protein of Neurospora, Wild Type (W) and Lysineless Mutant (L) In the L series 0.5 per cent of non-labeled lysine was added to the medium. The W series corresponds to the T series of Table II. The W and L series were cultivated simultaneously under the same conditions. T Isotope content of acetate Amino acids and degradation products Glycine, COOH.... a-carbon Slanine, COOH... N- and p-carbons... Valine, COOH... Residual carbon atoms.... Isoleucine, COOH.... and -,-carbons... Terminal CH,-y.... if CH,-s.... Proline, COOK... Residual carbon atoms.... Arginine, COOH... Lysine, COOH.... Residual carbon atoms. III 03 (CHs = 100) w L w (COOH = 100) L ously (IO, 11). Even here the distribution of isotope in different carbon atoms of the benzene ring and the side chain has been found analogous to the labeling of tyrosine isolated in similar experiments from Torulopsis and E. coli. The results presented in Tables I and II concerning the labeling patterns of individual amino acids from the normal strain of Neurospora do not reveal any marked deviations from what is known with regard to the acetate-amino acid interrelationship in other fungi. When the Zysineless mutant 4545 is grown under the same conditions as the wild type (with addition of a certain amount of exogenous lysine), the results differ very little in the two cultures (Table III). The 03 and Cl4

6 460 AMINO ACIDS IN NEUROSPORA labeling of respiratory carbon dioxide is approximately the same, as is the labehng of amino acid fragments and separate carbon atoms. The main difference is the isotope content of lysine, which in the case of the mutant is very low, indicating that the exogenous lysine has been incorporated as such into the protein of the mutant during the growth period. TABLE Isotope Content in Respiratory Carbon Dioxide jrorn Neurospora The wild type and the lysineless strain 4545 were cultivated on non-labeled acetate and uniformly P-labeled L-lysine. Cl4 activity is given in counts per minute per 15 mg. of barium carbonate, sampled at intervals. After 24 hours the isotope content in the mycelial material was determined. 2.5 mg. IV I-Lysine added per 50 ml. culture I 5.0 mg. Cl4 in respiratory CO2 I 15.0 mg. Wild type 55.5 zt 3.9 (O-S)* 93.1 f 1.5 (O-6)* 73.5 f 4.8 (O-S)* Lysineless 50.0 f 1.7 (G8) 68.1 f 2.1 (O-6) 66.7 f 3.0 (@8) Wild type 34.9 f 0.7 (8-16) 65.2 f 1.9 (6-12) 83.5 f 0.9 (8-16) Lysineless 29.5 f 1.4 (8-16) 50.6 f 0.7 (6-12) 75.5 f 2.5 (8-16) Wild type 30.7 f 0.8 (16-24) 60.6 f 1.1 (12-18) 215 f 16 (16-24) Lysineless 21.8 f 0.5 (16-24) 42.2 f 1.2 (12-18) 209 f 6 (16-24) Wild type Lysineless Cl4 in mycelium in 24 hrs. * The figures in parentheses represent time in hours. Experiments with Labeled Lysine Uniformly C14-labeled lysine, obtained by photosynthesis, was employed in three series of experiments in order to investigate the rate of transformation of lysine to carbon dioxide during the growth of the wild strain and the mutant A series of 300 ml. Erlenmeyer flasks containing 50 ml. of sterile acetate medium (non-labeled) plus varying amounts of labeled lysine was inoculated and incubated at 25 with shaking. A slow stream of air continuously swept out the respiratory carbon dioxide into a series of baryta traps. The first series of flasks contained 2.5 mg. of labeled lysine per flask, the second 5 mg., and the third 15 mg. One-half of the flasks of the different series were inoculated with conidia of the wild type and the other half with conidia from the lysineless strain. The respiratory carbon dioxide, trapped as barium carbonate, was sampled at intervals. After

7 ANDERSSON-KOTT6, EHRENSVXRD, HijGSTRijM, REIO, SALUSTE hours the experiment was terminated and the mycelium of each series was collected, washed, and dried. As is seen from Table IV, no appreciable difference was found between the mutant and the wild type with respect to the Cl4 content in the collected barium carbonate originating from the labeled lysine of the medium. The isotope content of the mycelial material was also of the same order in the two strains. DISCUSSION The results of the cultivation experiments with labeled acetate as the source of carbon clearly show that the pattern of isotope incorporation in different fragments of amino acids from wild type Neurospora has not been greatly changed by the mutation that is blocking the pathway leading to lysine. A detailed analysis might perhaps reveal minor differences caused by the interference of accumulated precursor material in the lysineless mutant. However, with the exception that the protein-bound lysine is non-labeled, the total picture of the mutant with regard to isotope incorporation in different amino acids is qualitatively the same as that of the wild type. One might expect from the above experiments that the ability of the mutant strain to incorporate exogenous lysine as such into protein should bring about marked differences with regard to the utilization of labeled lysine by the mutant compared to the wild type. However, the isotope content in the respiratory carbon dioxide and in the washed mycelium after termination of the experiments does not show any striking difference in the case of the two strains. This situation recalls the investigations of Abelson et al. (12) showing that an exogenous amino acid in the culture medium of E. coli will suppress one or several steps in the reaction series leading to the same amino acid. From our experiments it seems that we are dealing with the same phenomenon in wild type Neurospora, supplied with exogenous lysine, which incorporates this amino acid into protein at the same rate the lysineless mutant does. Further experiments on Neurospora of the type outlined by Abelson et al. might reveal the nature of the interference caused by the administration of exogenous amino acids to the medium. Since the carboxyl of lysine in the wild type is clearly derived from the acetic acid carboxyl, it would be expected that the sequence of reactions leading to lysine and its precursors should be influenced by the availability of active acetate and C4 fragments. For this reason a series of 50 ml. cultures, inoculated with lo6 conidia per ml., was harvested at intervals and the dry weight of the mycelia determined. The medium in one series was supplied with acetate as the sole source of carbon, in the other with sucrose,

8 462 AMINO ACIDS IN NEGROSPORA 750 mg. per culture. In the acetate series 2.5 WM of lysine per culture gave the same yield of mycelium with the wild type and the lysineless mutant; higher concentration than 5 PM decreased the yield considerably in the mutant. In the sucrose series, however, the yield rose steadily with increasing amounts of lysine, both in the wild type series and the mutant series. Thus, for Neurospora on acetate medium, concentrations higher than 5 pm of lysine are inhibitory, in contrast to the situation in sucrose medium. SUMMARY 1. Neurospora crassa has been cultivated on C13H&1400Na and the isotope content of the respiratory carbon dioxide and of different carbon atoms of amino acids determined. The general labeling pattern corresponded closely to that of Torulopsis utilis, and, with the exception of lysine, to that of Escherichia coli. A comparison of the incorporation of 03 and Cl4 from the labeled acetate into amino acids of the wild type Neurospora and the lysineless strain 4545 did not show any marked difference, except that lysine incorporated into the protein of the mutant originated wholly from the exogenous, non-labeled lysine present in the medium. Tartrate was not utilized for growth in the presence of acetate. 2. The extent of catabolism and utilization of uniformly C14-labeled lysine was of the same magnitude in the mutant and in the wild type, as determined from the Cl4 content of the mycelia and of the respiratory carbon dioxide. 3. With increasing amounts of exogenously supplied lysine the mutant strain cultivat.ed on acetate medium reaches an optimal value with regard to yield of mycelial material; concentrations of lysine higher than 100 PM per liter are inhibitory for both the mutant and the wild type. On sucrose medium a similar inhibitory effect is not observed. Our thanks are due to the Swedish State Scientific and Medical Councils, the Hierta-Retzius Foundation for Scientific Research, and the Kristiane and 0. F. Hedstriims Memorial Fund for financial support of this work. BIBLIOGRAPHY 1. Beadle, G. W., and Tatum, E. L., Am. J. Bot., 32, 678 (1945). 2. Raddiley, J., EhrensvBrd, G., Johansson, R., Reio, L., Saluste, E., and Stjernholm, R., J. Biol. &em., 183, 771 (1950). 3. Ehrensviird, G., Reio, L., Saluste, E., and Stjernholm, It., J. Biol. C/mm., 189, 93 (1951). 4. Cutinelli, C., Ehrensvgrd, G., Reio, L., Saluste, E., and Stjernholm, R., Acta them. Stand., 6, 353 (1951). 5. Cutinelli, C., Ehrensviird, G., Reio, L., Saluste, E., and Stjernholm, It., Ark. Kemi, 3, 315 (1951).

9 ANDERSSON-KOTT6, EHRENSV;iRD, HijGSTRijM, REIO, SSLUSTE Cutinelli, C., Ehrensvilrd, G., HBgstrijm, G., Reio, L., Saluste, E., and Stjernholm, R., Ark. Kemi, 3, 501 (1951). 7. HBgstriim, G., Acta &em. Stand., 7, 45 (1953). 8. Phares, E. F., Arch. Biochem. and Biophys., 33, 173 (1951). 9. Marvel, C. S., and Rands, R. D., J. Am. Chem. Sot., 72, 2642 (1950). 10. Reio, L., and Ehrensvkrd, G., Ark. Kemi, 6, No. 28, 301 (1953). 11. Ehrensvkd, G., and Reio, L., Ark. Kemi, 5, No. 20, 229 (1953). 12. Abelson, P. H., Bolton, E. T., and Aldous, E., J. Biol. Chem., 198, 173 (1952).

10 AMINO ACID FORMATION AND UTILIZATION IN NEUROSPORA I. Andersson-Kottö, G. Ehrensvärd, G. Högström, L. Reio and E. Saluste J. Biol. Chem. 1954, 210: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites 0 references, 0 of which can be accessed free at tml#ref-list-1